Polypeptide having hadh dependent hmf reductase activity

ABSTRACT

The invention relates to an isolated polypeptide having NADH dependent HMF reductase activity, wherein said polypeptide shows 80% homology to the amino acid sequence shown in SEQ ID NO:2 and which differs from SEQ ID NO:2 in that at least S117L and Y295 or S110 is substituted, a nucleotide sequence coding for said polypeptide, a vector comprising said polypeptide or nucleotide sequence, host comprising said nucleotide sequence or vector as well as the use of the polypeptide for the reduction of furan or carbonyl compounds in lignocellulosic material or in any furan or carbonyl containing material.

FIELD OF INVENTION

The invention relates to an isolated polypeptide having NADH dependentHMF reductase activity, wherein said polypeptide shows 80% homology tothe amino acid sequence shown in SEQ ID NO:2 and which differs from SEQID NO:2 in that at least S117L and Y295 or S110 are substituted, anucleotide sequence coding for said polypeptide, a vector comprisingsaid polypeptide or nucleotide sequence, host comprising said nucleotidesequence or vector as well as the use of the polypeptide for thereduction of furan or carbonyl compounds in lignocellulosic material orin any furan or carbonyl containing material.

BACKGROUND OF INVENTION

Bioethanol production from renewable feedstock by baker's yeastSaccharomyces cerevisiae has become an attractive alternative to fossilfuels. However, the availability of starch or sucrose based feedstocksuch as corn grain or sugar cane is expected to be insufficient to coverfuture worldwide needs for bioethanol (Gray et al., 2006. Bioethanol.Current Opinion Chemical Biology. 10(2):141-146). A foreseen solution isthe utilization of lignocellulosic feedstocks, such as corn stover,wheat straw, sugar cane bagasse, wood, etc (Hahn-Hägerdal et al., 2006.Bioethanol—the fuel of tomorrow from the residues of today. TrendsBiotechnol. 24(12):549-556). This requires overcoming new challengesassociated with the utilization of these complex raw materials.

One of these challenges concerns the presence of inhibitory compoundssuch as small aliphatic low molecular weight acids, furan derivatives,carbonyl compounds and phenolics that are released during thepretreatment and hydrolysis of lignocellulosic raw materials (Almeida etal., 2007. Increased tolerance of inhibitors in lignocellulosichydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol,82:340-349). Among these compounds, the presence of the furan derivative5-hydroxymethylfurfural (HMF), that originates from the dehydration ofhexoses, has been reported to result in reduced ethanol productivityduring the fermentation of lignocellulosic hydrolysates by S. cerevisiae(Taherzadeh et al. 2000. Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl Microbiol Biotechnol.53(6):701-708.). When compared in equimolar amounts with furfural,another inhibitory furan derivative found in hydrolysates, both thevolumetric ethanol productivity and the sugar consumption rate by thecells are lower with HMF (Larsson et al., 1999. The generation offermentation inhibitors during dilute acid hydrolysis of softwood.Enzyme Microbial Technology 24:151-159.). Therefore, either removing HMFand/or improving cellular HMF detoxification are crucial for anindustrial fermentation process based on lignocellulosic feedstocks.

WO03072602 discloses a polypeptide having Serin in position 295.However, the polypeptide lacks a leucine in position 117.

Nilsson et al., Applied and Environmetal Microbiology, December 2005,vol 71, page 7866-7871 disclosed that cell extracts from anlignocellulose hydrolysaste tolerant strain TMB3000 displayed apreviously unknown NADH-dependent HMF reducing activity, which was notpresent in the less tolerant strain CBS 8066.

An absolute requirement for the development of fermentation processesbased on lignocellulosic feedstocks is the development and optimisationof micro-organisms which are tolerant against the inhibiting compoundsmentioned above or strain that can utilise/degrade the inhibitingcompounds. S. cerevisiae strains have been shown to reduce HMF andfurfural (Larsson et al., 1999. The generation of fermentationinhibitors during dilute acid hydrolysis of softwood. Enzyme MicrobialTechnology 24:151-159) to 2,5-dimethanol (2,5-bis-hydroxymethylfuran)and 2-furanmethanol respectively, however the reduction rate is low andstrain dependent. Strain TMB3000 (Lindén et al., 1992. Isolation andcharacterization of acetic acid-tolerant galactose-fermenting strains ofSaccharomyces cerevisiae from a spent sulfite liquor fermentation plant.Applied Environmental Microbiology. 58(5):1661-1669) appears to be, sofar, the most tolerant strain that can grow in undiluted woodhydrolysates (Brandberg et al., 2005. Continuous fermentation ofundetoxified dilute acid lignocellulose hydrolysate by Saccharomycescerevisiae ATCC 96581 using cell recirculation. Biotechnology progress,21(4):1093-1101).

SUMMARY OF THE INVENTION

The invention relates to the isolation of a new polypeptide having NADHdependent HMF reductase activity, wherein said polypeptide has theability to reduce a number of compounds. One example of a polypeptideincludes a mutated alcohol dehydrogenase (ADH1) from Saccharomycescerevisiae, which in the native form cannot reduce HMF which whenmutated can reduce HMF. The invented polypeptide may be used in severalprocesses for the production of bulk and platform chemicals fromlignocellulosic material, such as lignocellulosic feedstock, whereinthere is a need to detoxify HMF or other furans or derivatives thereofor carbonyl compounds. Examples of biofuels, bulk and platform chemicalsinclude ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric,malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbicacid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glutaric acid, glutamic acid, itaconicacid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids,fatty-derived molecules, isoprenoids, isoprenoid-derived molecules,alkanes, isopentanol, isoamylacetate. When using the new polypeptide,increased specific productivity (gram product per gram cell and hour)will be achieved due to the faster removal of inhibitory furan compoundsand carbonyl compounds from the medium.

In a first aspect the invention relates to an isolated polypeptidehaving NADH dependent HMF reductase activity, wherein said polypeptideshows 80% homology to the amino acid sequence shown in SEQ ID NO:2 andwhich differs from SEQ ID NO:2 in that at least S117L and Y295 or S110are substituted. By the introduction of a single substitution into SEQID NO:2 and at the same time maintaining L in position an improvedpolypeptide is obtained which efficiently reduces HMF and furfural.

In a second aspect the invention relates to an isolated polypeptide asdefined above, wherein said polypeptide differs from SEQ ID NO:2 in thatat least Y295C or Y295S or Y295T or S110P are substituted

In a third aspect the invention relates to a nucleotide sequenceencoding a polypeptide as defined above.

In a fourth aspect the invention relates to a vector comprising thenucleotide sequence.

In a fifth aspect the invention relates to a host cell comprising thenucleotide sequence or the vector.

In a six aspect the invention relates to the use of the polypeptidehaving NADH dependent HMF reductase activity, the nucleotide sequence,the vector or the host cell for the production of bulk chemicals fromlignocellulosic feedstocks, such as those mentioned above. By the use ofthe improved polypeptides there is an increased sugar consumption rateand growth rate as well as if the host is used for the production ofethanol there is an increased rate of ethanol production by themicroorganism compared to when the native polypeptide is used.

By providing a new polypeptide as shown in SEQ ID NO:2 having NADHdependent HMF reductase activity it will for the first time be possibleto reduce furan compounds or carbonyl compounds or furan derivatives,such as 5-hydroxymethyl-2-furaldehyde (HMF) using NADH as a co-factor.HMF has been reported to reduce both cell growth and ethanolproductivity in baker's yeast, most commonly used for industrial ethanolproduction. These two problems will now be reduced or eliminated whenthe new isolated polypeptide is added to the process or the new isolatednucleotide sequence is transferred into a host cell, which is to be usedin a process where normally the problem with HMF is present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Substrate and HMF consumption and main product formation instrains TMB3280 (empty plasmid) (A, B) and TMB3206 (mutated ADH1) (C, D)under aerobic conditions. A, C=without HMF addition; B, D=with 20 mM HMFaddition. Symbols: ⋄ glucose, ∘ OD₆₂₀, x HMF, ▪ ethanol.

FIG. 2—Substrate and HMF consumption and main product formation instrains TMB3280 (empty plasmid) (A, B) and TMB3206 (mutated ADH1) (C, D)under anaerobic conditions. A, C=without HMF addition; B, D=with 20 mMHMF addition. Symbols: ⋄ glucose, ∘ OD₆₂₀, Δ HMF, ▪ ethanol.

FIG. 3—Fermentation profile of control strain TMB3280 (A) and strainTMB3206 overexpressing the mutated ADH1 gene from TMB3000 (B) in pulseexperiment with spruce hydrolysate. Strains are first grown in definedmedium, then spruce hydrolysate is added after 15-20 hours. Symbols:Furfural (⋄), HMF (Δ), Glucose (), Ethanol (▴), CO2 evolution rate orCER (-).

FIG. 4—Substrate and HMF consumption and ethanol formation in strainsTMB3290 (empty plasmid) (A) and TMB3291 (ADH1-S110P-Y295C) (B) underanaerobic conditions. Symbols: (▪) glucose, (▴) xylose, (⋄) HMF, (□)ethanol.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “nucleotide sequence” is intended to mean a consecutive stretchof three or more regions of nucleotide sequences. The nucleotides can beof genomic DNA, cDNA, RNA, semisynthetic or synthetic or a mixturethereof. The term includes single and double stranded forms of DNA orRNA.

The term “analogue thereof” is intended to mean that part of or theentire polypeptide of SEQ ID NO:2 is based on non protein amino acidresidues, such as aminoisobutyric acid (Aib), norvalinegamma-aminobutyric acid (Abu) or ornitihine. Examples of other nonprotein amino acid residues can be found athttp://www.hort.purdue.edu/rhodcv/hort640c/polyam/po00008.htm.

The term polypeptide “homology” is understood as the degree of identitybetween two sequences indicating a derivation of the first sequence fromthe second. The homology may suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, 443-453. The following settings for amino acid sequencecomparison are used: GAP creation penalty of 3.0 and GAP extensionpenalty of 0.1. The relevant part of the amino acid sequence for thehomology determination is the mature polypeptide.

The term “vector” as used herein refers to a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which is modified to contain segments of nucleic acidsin a manner that would not otherwise exist in nature. The term vector issynonymous with the term “expression cassette” when the vector containsthe control sequences required for expression of a coding sequence ofthe present invention. The term vector is also synonymous with the term“integration nucleic acid construct” or “integration fragment” when theconstruct is to be used to integrate the construct/fragment into thegenome of a host.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide encoding a polypeptide of the present invention. Eachcontrol sequence may be native or foreign to the nucleotide sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, polyadenylation sequence, pro-peptide sequence, promoter,and transcription terminator. At a minimum, the control sequencesinclude a promoter, and transcriptional and translational stop signals.The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

The term “host cell”, as used herein, includes any cell type which issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid.

In the present context, amino acid names and atom names are used asdefined by the Protein DataBank (PNB) (www.pdb.org), which is based onthe IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acidsand Pep-tides (residue names, atom names etc.), Eur J. Biochem., 138,9-37 (1984) together with their corrections in Eur J. Biochem., 152, 1(1985). The term “amino acid” is intended to indicate an amino acid fromthe group consisting of alanine (Ala or A), cysteine (Cys or C),aspartic acid (Asp or D), glutamic acid (Glu or E), phenyl-alanine (Pheor F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I),lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine(Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg orR), serine (Ser or S), threonine (Thr or T), valine (Val or V),tryptophan (Trp or W) and tyrosine (Tyr or Y), or derivatives thereof.

The terminology used for identifying amino acid positions is illustratedas follows: S110 indicates that the position 110 is occupied by a serineresidue in the amino acid sequence shown in SEQ ID NO:2. S110P indicatesthat the serine residue of position 110 has been substituted with aproline residue.

Polypeptide

The invention relates to a new polypeptide having NADH dependent HMFreductase activity, wherein said polypeptide shows 80% homology to theamino acid sequence shown in SEQ ID NO:2, at least 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or being thesame as SEQ ID NO:2. The invented polypeptide having some uniqueproperties since the enzyme uses NADH as a co-factor for the reductionof HMF and other furan compounds and carbonyl compounds such as thoseproduced during the processing of lignocellulosic material.

Another feature of the isolated polypeptide being that the amino acidresidue in position 295 or 110 of SEQ ID NO:2 is substituted. However,that particular amino acid may be altered by for example substitution toanother amino acid residue, such as a small uncharged amino acidresidue. Examples are Y295C or Y295S or Y295T or S110P.

In addition, the polypeptide shown in SEQ ID NO:2 the amino acid residueS117 should be substituted to L. Substitution of the amino acid residuepresent in position 295 being important and increases the furfuralreductase and substitution of both the amino acid residue present inposition 295 and 110 for the production of ethanol.

One example of a polypeptide is shown in SEQ ID NO:2, in which the aminoacid residues 295 is C or S, 110 is P. Another specific example is whenthe amino acid residue 59 is T, 210 is P, 148 is E, 152 is V and 295 isC or S of SEQ ID NO:2. The polypeptide having a new activity, i.e.,having NADH dependent HMF reductase activity can therefore be used in aprocess in which there is a need of detoxifying furan compounds andcarbonyl compounds. There will also be an increased specificproductivity due to that there will be a faster reduction of the furansand carbonyl compounds, the toxic effects of which slow down the wholeprocess. The polypeptide may be synthetic partly or completely as longas the activity remains.

The polypeptide may also be a hybrid polypeptide. The term “hybridenzyme” or hybrid polypeptide” is intended to mean for example thosepolypeptides of the invention that comprises a first set of amino acidsequences comprising the amino acid residues from about 110 to about 295as shown in SEQ ID NO:2 fused/linked to a second set of amino acidresidues and thereby producing a completely synthetic nucleotidesequence based on the knowledge on suitable amino acid sequences, suchas linkers, homologous amino acid sequences etc.

The invented polypeptide was cloned from a yeast strain, Saccharomycescerevisiae TMB3000 (ATCC96581) which was shown to have a unique NADHdependent HMF reductase activity. During the analysis of the polypeptideit was found that the polypeptide was similar to the ADH1 polypeptide,which normally cannot reduce HMF. The sole introduction of a fewmutations within the polypeptide altered the activity of the polypeptideand it was surprisingly found that the polypeptide had NADH dependentHMF reductase activity. However, the polypeptide may be obtained fromany source or even synthetically made as long as the polypeptide has theproperties as the defined polypeptide.

Nucleotide Sequence

Another object of the invention relates the nucleotide sequence shown inSEQ ID NO:1 or by a nucleotide sequence having from at least 60, 65, 70,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% homology to the nucleotide sequence shown in SEQ ID NO:1encoding the polypeptide having NADH dependent HMF reductase activity.The nucleotide sequence may be obtained by standard cloning proceduresused in genetic engineering to relocate the DNA sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desired DNAfragment comprising the DNA sequence encoding the polypeptide ofinterest, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the DNA sequence will be replicated. An isolated DNAsequence may be manipulated in a variety of ways to provide forexpression of the polypeptide of interest. Manipulation of the DNAsequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying DNA sequences utilizing recombinant DNA methods are well knownin the art.

The nucleotide sequence to be introduced into the DNA of the host cellmay be integrated in vectors comprising the nucleotide sequence operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. A nucleotide sequence encoding a polypeptidemay be manipulated in a variety of ways to provide for expression of thepolypeptide

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofthe nucleotide sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including native, mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell. The promoter may be a weak or a strongpromoter that is constitutive or regulated in the host to be used.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in bacteria of the present invention aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook J et al., 1989 MolecularCloning. A Laboratory Manuel. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters may be obtained for example from thegenes for Saccharomyces cerevisiae enolase (ENO1), S. cerevisiaegalactokinase (GAL1), S. cerevisiae alcohol dehydrogenase 2 (ADH2), S.cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH1), S.cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) (Bitter andEgan. Expression of heterologous genes in Saccharomyces cerevisiae fromvectors utilizing the glyceraldehyde-3-phosphate dehydrogenase genepromoter. (1984) Gene 32: 263-274, S. cerevisiae alcohol dehydrogenase 1(ADH1), S. cerevisiae 3-phosphoglycerate kinase (PGK1) or S. cerevisiaecytochrome C (CYC1) (Karhumaa et al. Investigation of limiting metabolicsteps in the utilization of xylose by recombinant Saccharomycescerevisiae using metabolic engineering. (2005) Yeast 5:359-68). Anotherexample of a yeast promoter is the constitutive truncated HXT7 promoter(Hauf et al. Enzym Microb Technol (2000) 26:688-698). Other suitablevectors and promoters for use in yeast expression are further describedin EP A-73,657 to Hitzeman, which is hereby incorporated by reference.

The present invention also relates to vectors as defined above which maycomprise a DNA sequence encoding the polypeptide, a promoter, andtranscriptional and translational stop signals as well as other DNAsequences. The vector comprises various DNA and control sequences knownfor a person skilled in the art, which may be joined together to producea vector which may include one or more convenient restriction sites toallow for insertion or substitution of the DNA sequence encoding thepolypeptide at such sites. Alternatively, the DNA sequence of thepresent invention may be expressed by inserting the DNA sequence or aDNA construct comprising the sequence into an appropriate vector forexpression. In creating the vector, the coding sequence is located inthe vector so that the coding sequence is operably linked with theappropriate control sequences for expression and possibly secretion.

The vector may be any vector (e.g., a plasmid, virus or an integrationvector), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the DNA sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, a cosmid or an artificial chromosome. The vector maycontain any means for assuring self-replication. Alternatively, thevector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. The vector system maybe a single vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon. The vector may also be an integration vectorcomprising solely the gene or part of the gene to be integrated.

The vectors of the present invention preferably contain one or moreselectable markers, which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophsand the like.

Useful expression vectors for eukaryotic hosts, include, for example,vectors comprising control sequences from SV40, bovine papilloma virus,adenovirus and cytomegalovirus. Specific vectors are, e.g.,pcDNA3.1(+)Hyg (Invitrogen, Carlsbad, Calif., U.S.A.) and pCI-neo(Stratagene, La Jolla, Calif., U.S.A.). Useful expression vectors foryeast cells include, for example, the 2μ (micron) plasmid andderivatives thereof, the YIp, YEp and YCp vectors described by Gietz andSugino (1988, “New yeast vectors constructed with in vitro mutagenizedyeast genes lacking six-base pair restriction sites”, Gene 74:527-534),the vectors described in Mumberg et al (Mumberg, Muller and Funk, 1995,“Yeast vectors for the controlled expression of heterologous proteins indifferent genetic backgrounds.” Gene 156:419-422), YEplac-HXT vector(Karhumaa et al., 2005. Investigation of limiting metabolic steps in theutilization of xylose by recombinant Saccharomyces cerevisiae usingmetabolic engineering. Yeast. 22(5):359-68)), the POT1 vector (U.S. Pat.No. 4,931,373), the pJSO₃₇ vector described in Okkels, Ann. New YorkAcad. Sci. 782, 202-207, 1996, the pPICZ A, B or C vectors (Invitrogen).Useful vectors for insect cells include pVL941, pBG311 (Cate et al.,“Isolation of the Bovine and Human Genes for Mullerian InhibitingSubstance and Expression of the Human Gene in Animal Cells”, Cell, 45,pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both available fromInvitrogen). Useful expression vectors for bacterial hosts include knownbacterial plasmids, such as plasmids from E. coli, including pBR322,pET3a and pET12a (both from Novagen Inc., Wis., U.S.A.), wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages. Examples of suitable viralvectors are Adenoviral vectors, Adeno associated viral vectors,retroviral vectors, lentiviral vectors, herpes vectors and cytomegaloviral vectors.

The vectors of the present invention may contain an element(s) thatpermits stable integration of the vector into the host cell genome orautonomous replication of the vector in the cell independent of thegenome of the cell. The vectors of the present invention may beintegrated into the host cell genome when introduced into a host cell.For integration, the vector may rely on the DNA sequence encoding thepolypeptide of interest or any other element of the vector for stableintegration of the vector into the genome by homologous or nonhomologous recombination.

Alternatively, the vector may contain additional DNA sequences fordirecting integration by homologous recombination into the genome of thehost cell. The additional DNA sequences enable the vector to beintegrated into the host cell genome at a precise location(s) in thechromosome(s). To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of nucleotides, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding DNA sequences. Onthe other hand, the vector may be integrated into the genome of the hostcell by non-homologous recombination. These DNA sequences may be anysequence that is homologous with a target sequence in the genome of thehost cell, and, furthermore, may be non-encoding or encoding sequences.More than one copy of a DNA sequence encoding a polypeptide of interestmay be inserted into the host cell to amplify expression of the DNAsequence.

An Isolated Host Cell

The invention also relates to an isolated host cell, which comprises thenucleotide sequence as defined above either in a vector, such as anexpression vector or alternatively has the nucleotide sequenceintegrated into the genome, e.g. by homologous or heterologousrecombination. The nucleotide sequence may be present as a single copyor multiple copies.

The host cell may be any appropriate prokaryotic or eukaryotic cell,e.g., a bacterial cell, a filamentous fungus cell, a yeast, a plant cellor a mammalian cell. Any suitable host cell may be used for themaintenance and production of the vector of the invention, such as aneukaryotic or prokaryotic cell, for example bacteria, fungi (includingyeast), plant, insect, mammal, or other appropriate animal cells or celllines, as well as transgenic animals or plants. The host cell may be ahost cell belonging to a GMP (Good Manufacturing Practice) certifiedcell-line, such as a mammalian cell-line.

Examples of bacterial host cells include Escherichia coli, Zymomonas sp.and Klebsiella sp.

Examples of suitable filamentous fungal host cells include Aspergillussp., e.g. A. oryzae, A. niger, or A. nidulans, Fusarium sp. or Hypocrea(formerly Trichoderma) sp.

Examples of suitable yeast host cells include Saccharomyces sp., e.g. S.cerevisiae, S. bayanus or S. carlsbergensis, Schizosaccharomyces sp.such as Sch. pombe, Kluyveromyces sp. such as K. lactis, Pichia sp. suchas P. stipitis, P. pastoris or P. methanolica, Hansenula sp., such as H.polymorpha, Candida sp., such as C. shehatae or Yarrowia sp. Examples ofS. cerevisiae strains are DBY746, AH22, S150-2B, GPY55-15Bα, CEN.PK,USM21, TMB3500, TMB 3400, VTT-A-63015, VTT-A-85068, VTT-c-79093) andtheir derivatives as well as Saccharomyces sp. 1400, 424A (LNH-ST), 259A(LNH-ST) and derivatives thereof.

Examples of suitable insect host cells include a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioani cells(High Five) (U.S. Pat. No. 5,077,214).

Examples of suitable mammalian host cells include Chinese hamster ovary(CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines(COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells(e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 orATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well asplant cells in tissue culture.

The invented polypeptide, nucleotide sequence, vector or host cell maybe used in the production of biofuels, bulk and platform chemicals, suchas include ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric,malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbicacid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glutaric acid, glutamic acid, itaconicacid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids,fatty-derived molecules, isoprenoids, isoprenoid-derived molecules,alkanes, isopentanol, isoamylacetate. The process concept for theconversion of lignocellulosic feedstock to a bulk chemical such asethanol may include steps such as a pre-treatment or fractionation stepin which the chopped raw material is exposed to neutral, acidic oralkaline pH, at high temperature with or without air/oxygen added, sothat the hemicellulose fraction is partially hydrolysed to monomeric andoligomeric sugars, rendering the cellulose fraction susceptible forhydrolysis or in which the chopped raw material is exposed to an organicsolvent such as acetone, ethanol or similar, at high temperature, sothat the lignin fraction is dissolved and extracted rendering thecellulose and hemicellulose fraction susceptible to hydrolysis. Thehydrolysis of the pretreated and fractionated material may be performedwith concentrated or diluted acids or with cellulolytic andhemicellulolytic enzyme mixtures.

Following examples are intended to illustrate, but not to limit, theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1 Identification of NADH-Dependent HMF ReductasePurification of NADH-Dependent HMF Reductase

The industrial strain TMB3000 (ATCC96581) (Lindén et al., 1992,Isolation and characterization of acetic acid-tolerantgalactose-fermenting strains of Saccharomyces cerevisiae from a spentsulfite liquor fermentation plant. Applied Environmental Microbiology58(5):1661-1669) was grown in rich medium containing 10 g/l yeastextract, 20 g/l peptone and 20 g/l glucose supplemented with 10% sprucehydrolysate (Nilvebrant et al., 2003, Limits for alkaline detoxificationof dilute-acid lignocellulose hydrolysates. Applied BiochemistryBiotechnology. 105-108:615-628) adjusted to pH 5.5. The hydrolysate wascentrifuged for 10 minutes at 5000 g and supernatant was injectedthrough 2μ filter (Sarstedt, Nümbrecht, Germany) before addition to theautoclaved medium. Growth took place in 1 L shake flasks (non-baffled),containing 250mL medium at 200 rpm and 30° C. Inoculation from a 5 mlover-night pre-culture in rich medium (without hydrolysate) was to OD620of 0.1, and cells were harvested at OD620 about 4, after 24 hours.

Cells were harvested and washed twice with double distilled water beforebeing re-suspended in Y-PER detergent (Pierce, Rockford, Ill.), 1 ml/0.6g cells. After gentle shaking at room temperature for 50 minutes thesuspension was centrifuged for 20 minutes at 15000 g. Supernatant wascollected and used for enzymatic assays and further purification steps.Coomassie protein assay reagent (Pierce, Rockford, Ill.) was used inBradford assay for the determination of protein concentration accordingto the manufacturer's recommendations. Measurements of NADH-dependentfuran reduction were conducted according to Wahlbom and Hahn-Hägerdal(2002, Furfural, 5-hydroxymethyl furfural, and acetoin act as externalelectron acceptors during anaerobic fermentation of xylose inrecombinant Saccharomyces cerevisiae. Biotechnology and Bioengineering.78(2):172-178) with the following modification: NADH concentration wasset to 200 μM. All measurements were performed at 30° C. in a U-2000spectrophotometer (Hitachi, Tokyo, Japan).

For the identification of the enzyme responsible for NADH-dependent HMFreduction, the protein solution was fractionated using ammonium sulphateprecipitation, size exclusion and affinity chromatography according tothe following protocol: cell extract (15 mL) was mixed with 10 mlsaturated ammonium sulphate solution to a final concentration of 40%.After 1 hour of gentle shaking at 4° C. the mixture was centrifuged at15000 g for 20 minutes. The pellet was kept on ice for immediate usageor at −20° C. for later usage (named 40% pellet), while the supernatantwas mixed with an equal volume that yielded a 70% ammonium sulphatesolution. After 3 hours of gentle shaking at 4° C., centrifugation wasrepeated at 15000 g for 20 minutes. The pellet was again kept on ice orat −20° C. (named 70% pellet), while the supernatant (˜40 mL) wasdialyzed over-night against 12 l distilled water at 4° C. usingSpectra/Por membrane tubing (MWCO 12 kDa-14 kDa) from SpectrumLaboratories, Calif., USA. Size exclusion chromatography was performedusing HiLoad 16/60 Superdex 200 column (Amersham Pharmacia Biotech,Uppsala, Sweden). Buffer contained 100 mM Tris-HCl and 0.5M NaCl (pHraised to 6.7 with NaOH), both from Merck, Darmstadt, Germany. Flow ratewas adjusted to 1 ml/min using a FPLC system (Amersham PharmaciaBiotech, Uppsala, Sweden) governed by LCC-501 Plus controller. 50 ml ofswollen Red Sepharose CL-6B were packed in XK50 column, both fromAmersham Pharmacia Biotech, Uppsala, Sweden. Binding buffer contained 20mM Tris-HCl, pH 6.4, 5 mM MgCl₂, 0.4 mM EDTA and 2 μM β-mercaptoethanol.Elution buffer contained in addition 10 mM β-NAD₊ (Sigma-Aldrich, St.Louis, Mo., USA). Flow rate was adjusted to 5 ml/min using the FPLCsystem mentioned above.

Results from the purification steps are presented in Table 1.

TABLE 1 Purification of NADH-dependent HMF reductase from S. cerevisiaeTMB3000. Purification level is defined as the ratio between the specificactivity of a given step and the specific activity at the first step. 1unit (U) is defined as 1 μmol of NADH oxidized per min at 30° C. and pH6.7. Specific Total Total activity protein activity (mU/mg YieldPurification Step (mg) (units) protein) (%) level Crude extract 27965500 235 100 1 Ammonium 145 43935 303 67 1.28 sulfate precipitationSize exclusion 2.566 2065 805 3.15 3.4 chromatography Affinity 0.18 9325180 1.42 22 chromatography

Purified fractions were run on SDS-PAGE using Tris-HCl 4%-15% gradientprecast gel and Mini-PROTEAN 3 electrophoresis unit, both from Bio-Rad(Hercules, Calif., USA). Precision Plus Protein dual color (Bio-Rad,Hercules, Calif., USA) was used as standard. Gel staining was conductedusing 0.25% Coomassie brilliant blue R-250 (ICN Biomedicals, Aurora,Ohio, USA) in a 6:3:1 mixture of H₂O:MeOH:HAc. All mass-spectrometryresults were obtained from SWEGENE proteoinics resource centre in Lund,Sweden.

The final purified fraction showed one band on SDS-PAGE corresponding toa molecular mass of 37 kDa. The purified protein was identified as S.cerevisiae alcohol dehydrogenase 1 (Adh1) using electrospray ionizationmass spectrometry (ESI-MS).

Cloning of ADH1 Gene from S. cerevisiae TMB3000

The ADH1 gene from TMB3000 was amplified by PCR from genomic DNA andcloned into YEplac-HXT vector carrying strong constitutive truncated HXTpromoter (Hauf et al., 2000. Simultaneous genomic overexpression ofseven glycolytic enzymes in the yeast Saccharomyces cerevisiae. EnzymeMicrob Technol, 26: 688-698) (Karhumaa et al., 2005. Investigation oflimiting metabolic steps in the utilization of xylose by recombinantSaccharomyces cerevisiae using metabolic engineering. Yeast.22(5):359-368).

More specifically, genomic DNA of TMB3000 was extracted using Y-DER kit(Pierce, Rockford, Ill.), according to manufacturer's recommendations.PCR for the amplification of the ADH1 gene from TMB3000 was conductedusing Pwo DNA polymerase (Roche Diagnostics AB, Bromma, Sweden) with anannealing temperature of 42° C. Two forward and two reverse primers wereused in all possible combinations (restriction sites for later cleavageand ligation are underlined): ForwardA-5′-GGGCGGATCCATACAATGTCTATCCCAGAAA-3′, ForwardB-5′-GGGGGGATCCATGTCTATCCCAGAAACTC-3°, ReverseA-5′-CTTTAGATCTTTATTTAGAAGTGTCAACAACG-3′, ReverseB-5′-CTTTAGATCTGCTTATTTAGAAGTGTCAACA-3°. The purified amplicons weremixed and cleaved with BamHI and BglII (Fermentas, Vilnius, Lithuania),then used for ligation with plasmid YEplac-HXT that was previouslycleaved with the same restriction enzymes and de-phosphorylated. Theligation product was used to transform Escherichia coli DH5α (LifeTechnologies, Rockville, Md., USA) according to the method of Inoue etal. (1990, High efficiency transformation of Escherichia coli withplasmids. Gene 96:23-28). Transformants were selected on Luria-Bertani(LB) agar plates (Ausubel et al., 1995. Current protocols in molecularbiology. Wiley, New York) containing ampicillin (50 μg/mL). PCR wasperformed on several clones in order to verify correct ligation andplasmid was extracted to transform S. cerevisiae strain BY4741 ΔADH1(Invitrogen, Groningen, the Netherlands) using the lithium acetatemethod (Gietz et al., 1992. Improved method for high efficiencytransformation of intact yeast cells. Nucleic Acids Research20(6):1425). Transformants were selected anaerobically on SD-ura plates(Ausubel et al., 1995. Current protocols in molecular biology. Wiley,New York) containing 20 mM HMF (Sigma-Aldrich, location), 400 μg/ml ofTween 80 and 10 μg/l ergosterol. Plasmid was extracted from clonesshowing the highest NADH-dependent HMF reductase activity by growing thecells overnight in 5 ml SD medium without uracil, washing with doubledistilled water and resuspending in 1 ml protoplasting solution (1.2Msorbitol, 100 mM Tris pH7.5, 10 mM CaCl₂, 4 U/ml zymolyase, 0.5%(3-mercaptoethanol). After 30 minutes shaking at 30° C., cells wereharvested and used for plasmid extraction using the QIAprep SpinMiniprep Kit (Qiagen, Hilden, Germany). Plasmid was transformed to E.coli and selected on LB plates with 50 μg/ml ampicillin. Colonies weregrown in 5 mL LB medium containing ampicilin (50 μg/ml) and used forplasmid extraction using the QIAprep Spin Miniprep Kit (Qiagen, Hilden,Germany). The resulting plasmid was named YEplac-HXT-ADH1-mut.

ADH1 gene from YEPlac-HXT-ADH1-mut was sequenced using the Abi-PrismBigDye cycle sequencing kit (Applied Biosystems, Weiterstadt, Germany).Six mutations resulting in amino acid substitutions (Table 2) were foundfrom the comparison of the sequenced ADH1 gene from TMB3000 with thenative ADH1 gene (as reported in SGD, Saccharomyces genome database(SGD), www.yeastgenome.org, systematic name YOL086C).

TABLE 2 Comparison of native Adh1 amino acid sequence with predictedsequence of mutated adh1 from TMB3000. Position 59 110 117 148 152 295Native Adh1 V S L Q I Y Adh1 from TMB3000 T P S E V C

Example 2 In Vivo HMF Uptake with Strain Expressing NADH-DependentReductase

Overexpression in S. cerevisiae CEN.PK 113-5D

Plasmid YEplac-HXT-ADH1-mut encoding the mutated ADH1 gene (as describedin example 1), as well as the corresponding empty plasmid YEplacHXT(Karhumaa et al., 2005, Investigation of limiting metabolic steps in theutilization of xylose by recombinant Saccharomyces cerevisiae usingmetabolic engineering. Yeast. 22(5):359-368) were used to transform S.cerevisiae CEN.PK 113-5D (SUC2, MAL2-8c, MEL, ura3), resulting instrains TMB3206 and TMB3280, respectively. As expected, NADH-dependentHMF reductase activity was only detected in the strain carrying themutated ADH1 gene (data not shown).

Aerobic In Vivo HMF Reduction

Strains TMB3206 and TMB3280 were pre-grown overnight in 5 ml definedmedium (Verduyn et al., 1992. Effect of benzoic acid on metabolic fluxesin yeasts: a continuous-culture study on the regulation of respirationand alcoholic fermentation. Yeast, 8, 501-517) at 30° C. and 200 rpm.Cells were used for inoculation to OD620 nm=0.1 in 100 ml defined mediumsupplemented with 40 g/l glucose in 1 L—baffled shake flasks.Experiments, that were run at 30° C. and 200 rpm, were conducted withand without HMF (2 g/l) added for both strains. Samples were taken every2 to 4 hours.

Cell concentration was determined from absorbance measurements at 620 nmcalibrated against dry-weight measurements from duplicate samples. Fordry-weight samples 10 ml of the cell suspension were vacuum filteredthrough a pre-weighed Gelman filters (ø47 mm Supor-450, 0.45 μm).Filters were washed with water and dried. Samples for analysis ofmetabolite concentrations were taken regularly from the reactor. Thesamples were filtered through 0.2 μm filters. The concentrations ofglucose, ethanol and HMF were measured on an Aminex HPX-87H column(Bio-Rad, Hercules, Calif.) at 65° C. The mobile phase was 5 mM H₂SO₄with a flow rate of 0.6 ml/min. All compounds were detected with arefractive index detector, except for HMF which was detected with aUV-detector at 210 nm.

A representative experimental result is shown in FIG. 1. The glucoseconsumption, ethanol formation and biomass production were similar forboth strains when no HMF was added to the medium (FIG. 1A, 1C). On thecontrary, when adding HMF to the medium, considerably higher glucoseconsumption rate, ethanol and biomass production rate were observed instrain TMB3206 overexpressing the mutated ADH1 gene, as compared to thecontrol strain TMB3280 (FIG. 1B, 1D). The experiment confirmed thatincreased in vitro NADH-dependent HMF reductase activity correlated withincreased in vivo HMF reduction rate for strain TMB3206 overexpressingthe mutated ADH1 gene.

Anaerobic In Vivo HMF Reduction

Inoculum cultures of strains TMB3280 (control) and TMB3206(overexpressing the mutated ADH1 gene) were grown for 24 hours at 30° C.and 150 rpm in 1 L—shake-flasks with 100 ml of 2 times concentrateddefined mineral medium (as described in Verduyn et al., 1992, Effect ofbenzoic acid on metabolic fluxes in yeasts: a continuous-culture studyon the regulation of respiration and alcoholic fermentation. Yeast 8,501-517) supplemented with 40 g/l glucose and 200 ml/l phtalate buffer(10.2 g/L KH phtalate, 2.2 g/l KOH). The same defined medium, with orwithout HMF (2 g/l), was used in subsequent batch experiments. Themedium was supplemented with 60 g/l glucose, ergosterol (0.075 g/l) andTween 80 (0.84 g/l). The starting OD620 in batch fermentation was 0.5.Fermentation was carried out in 1 L media at 30° C. in Braun Biotechfermenters. Anaerobic conditions were maintained by continuouslysparging 0.2 litre/minute nitrogen gas. pH 5.5 was maintained with 3MKOH. The stirring rate was 200 rpm.

A representative experimental result for the two strains in the presenceand absence of HMF is shown in FIG. 2.

Both strains displayed slower growth, glucose conversion and ethanolproduction when HMF was added to the medium. However, HMF was convertedmuch faster with strain TMB3206 overexpressing mutated ADH1 gene thanwith the control strain TMB3280. Consequently, glucose consumptionstarted earlier in TMB3206 and full conversion of glucose to ethanol,biomass and other minor products was achieved within 35 hours (FIG. 2D).On the contrary, it took more than 45 hours for the control strainTMB3280 to reach a similar profile (FIG. 2B).

Example 3 Site-Directed Mutagenesis on NADH-Dependent HMF Reductase

Plasmid YEplacHXT-ADH1-mut encoding the mutated ADH1 gene from TMB3000(as described in example 1) as well as plasmid YEPlac-HXT encoding thenative ADH1 gene from CEN.PK 113-5D (named YEplacHXT-ADH1-nat) were usedfor site directed mutagenesis in order to investigate the influence ofvarious amino acid mutation on NADH-dependent furan reductase activity.

Site-directed mutagenesis was performed according to the followingprotocol: A two-step PCR was used for the generation of an ADH1 genewith the chosen amino-acid changes. Six primers were designed forreverse mutagenesis of the mutated ADH1 gene at position 110 (P110S),117 (S117L) and 295 (C295Y) and four other primers were designed for themutagenesis of the native ADH1 gene at position 110 (S110P) and 295(Y295C) (Table 3). Two primers for the amplification of ADH1 gene(mutated or not) were also created: primer ADH1 sense(5′-GGGGGGATCCATGTCTATCCCAGAAACTC-3′), carrying BamHI site and primerADH1 antisense (5′-CTTTAGATCTTTATTTAGAAGTGTCAACAACG-3′) carrying BglIIsite. For reverse mutation of the ADH1 gene from TMB3000, plasmidYEplacHXT-ADH1-mut was used as template. For mutation of the native ADH1gene, plasmid YEplacHXT-ADH1-nat was used as template.

TABLE 3 Primers used for the site-directed mutagenesis of native andmutated ADH1 gene. Primer name Sequence (5′-3′) Function P110S senseGTGAATTGGGTAACGAATCCAA Reverse mutation CTGTCCTCACGC at position 110P110S GCGTGAGGACAGTTGGATTCGT Reverse mutation antisense TACCCAATTCAC atposition 110 S117L sense CTGTCCTCACGCTGACTTGTCT Reverse mutationGGTTACACCCAC at position 117 S117L GTGGGTGTAACCAGACAAGTCA Reversemutation antisense GCGTGAGGACAG at position 117 C295Y senseCTCCATTGTTGGTTCTTACGTC Reverse mutation GGTAACAGAGCTG at position 295C295Y CAGCTCTGTTACCGACGTAAGA Reverse mutation antisense ACCAACAATGGAG atposition 295 S110P sense GGTAACGAACCCAACTGTC Create mutation at position110 S110P GACAGTTGGGTTCGTTACC Create mutation antisense at position 110Y295C sense TTGGTTCTTGCGTCGGTAAC Create mutation at position 295 Y295CGTTACCGACGCAAGAACCAA Create mutation antisense at position 295

In the first round of PCR, two fragments were amplified for eachtargeted mutation: one fragment using primer ADH1 sense and theantisense primer corresponding to the targeted mutation, and anotherfragment using primer ADH1 antisense and the sense primers correspondingto the targeted mutation. Pwo DNA polymerase (Roche Diagnostics AB,Bromma, Sweden) was used for amplification with the followingconditions: initial denaturation at 94° C. for 5 minutes then 30 cyclesconsisting of a denaturation step at 94° C. for 30 seconds, an annealingstep at 55° C. for 30 seconds and an elongation step at 72° C. for 1minute. The two amplified fragments were purified and used for thesecond round of PCR, together with primers ADH1 sense and ADH1antisense, Pwo DNA polymerase and at the following conditions: initialdenaturation at 94° C. for 5 minutes then 30 cycles consisting of adenaturation step at 94° C. for 30 seconds, an annealing step at 55° C.for 30 seconds and an elongation step at 72° C. for 1 minute. Theamplified fragment was purified and cleaved with BamHI and BglII(Fermentas, Vilnius, Lithuania). The cleaved PCR fragment was ligated todouble cleaved YEplacHXT using T4 DNA ligase (Fermentas, Vilnius,Lithuania). The ligation product was used to transform Escherichia coliDH5α cells according to Inoue et al. (1990. High efficiencytransformation of Escherichia coli with plasmids. Gene 96:23-28).Transformants were selected on LB medium with 50 μg/ml ampicillin andchecked for correct mutation and sequence by restriction analysis andDNA sequencing. Multiple mutations were performed sequentially.

Plasmid carrying individual or combined mutations within the ADH1 genewere introduced in strain CEN.PK 113-5D by transformation according tothe lithium acetate method (Gietz et al., 1992, Improved method for highefficiency transformation of intact yeast cells. Nucleic Acids Research20:1425). Selection was performed on SD plates without uracil. Theresulting strains were grown aerobically at 30° C. in 25 mL SD mediumwithout uracil (in 250 mL baffled flask). Cell extracts were prepared byharvesting the cells at OD₆₂₀ 7.5-8.5, re-suspending the cells in Y-PERsolution (Pierce, Rockford, Ill., USA), 1 mL/0.6gr cells, shaking atroom temperature for 50 min. The supernatant was collected aftercentrifugation for 20 min at 15000 g. In vitro NADH-dependent furanreductase activity was measured by following the oxidation of NADH at340 nm in a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan).The assay consisted of 10 mM furaldehyde (HMF or furfural), 200 μM NADHand cell extract in 100 mM phosphate buffer (pH 6.7) at 30° C. Resultsare presented in Table 4.

TABLE 4 Mutated amino acids and corresponding NADH-dependent HMF andfurfural in vitro activities. NADH-dependent Activity HMF FurfuralMutations reduc- Reduc- V59T Q148E I152V S110P L117S Y295C tase tase − −− + − − − ++ − − − − − + + +++ − − − + − + ++ ++ + + + − + + + + + + + +− + ++ ++ + + + + + − − + + + + − − + + +++ + + + + + + + + — nodetectable activity, + detectable activity (ranging from moderate (+) tovery high (+++)).

Example 4 Fermentation of Lignocellulosic Hydrolysate with StrainOverexpressing NADH-Dependent HMF Reductase

Strains TMB3280 (control) and TMB3206 (overexpressing mutated ADH1 genefrom TMB3000) were compared in anaerobic fermentation of undetoxifiedhydrolysate originating mainly from spruce in a two-stage dilute-kidhydrolysis process using sulphuric acid as the catalyst (Larsson et al.Development of a Saccharomyces cerevisiae strain with enhancedresistance to phenolic fermentation inhibitors in lignocellulosehydrolysates by heterologous expression of laccase. Appl EnvironMicrobiol 2001, 67:1163-1170).

Experiments were carried out by adding a single pulse of lignocellulosehydrolysate, previously adjusted to pH 5 with 6 M NaOH, to exponentiallygrowing cells in batch. More specifically, cells were pre-grown in 300ml shake flasks at 160 rpm and 30° C. for 24 h. The liquid volume in theprecultures was 100 ml with a glucose concentration of 15 g/l.Subsequent fermentation was inoculated with 6 ml of the preculture andcarried out in Belach BR 0.5 fermentors (Belach Bioteknik AB, Solna,Sweden) at 30° C. and a stirrer speed of 600 rpm. The initial workingvolume was 300 ml and the pH was kept constant at 5.0 by addition of0.75 NaOH. The fermentor was continuously sparged with 300 ml/minnitrogen gas (containing less than 5 ppm oxygen) to give anaerobicconditions, controlled by a mass flow meter (Bronkhurst Hi-Tec, Ruurlo,The Netherlands).

Initial concentrations of medium components were 2.67 times highercompared to the inoculum cultures in order to compensate for thedilution. Experiments were started by growing cells on 30 g of glucosein 300 ml growth medium. Next, a single addition of 300 ml ofhydrolysate was made when the biomass concentration reached approx. 4g/l monitored by OD₆₁₀ measurements. The carbon dioxide evolution ratewas monitored on-line by measuring the concentrations of carbon dioxideand oxygen in the outgoing gas from the reactor with a CP460 gasanalyser (Belach Bioteknik AB, Solna, Sweden). The gas analyzer wascalibrated using a gas containing 20% oxygen and 5% carbon dioxide. Cellconcentration was determined from absorbance measurements at 610 nm anddry-weight measurements were made from duplicate 10 ml samples, whichwere centrifuged, washed with distilled water and dried for 24 h at 105°C. Samples for metabolite measurement were immediately centrifuged,filtered through 0.2 μm filters and stored at −20° C. until analysis.The concentrations of glucose, mannose, galactose, HMF and furfural weremeasured on an Aminex HPX-87P column (Bio-Rad, USA) at 85° C., elutedwith ultra-pure water at 0.6 ml/min. The concentrations of ethanol wasmeasured on an Aminex HPX-87H column (Bio-Rad, USA) at 60° C. The eluentused was 5 mM H2SO4 at a flow rate of 0.6 ml/min. Compounds weredetected with a refractive index detector, except for HMF and furfuralwhich were detected with a UV-detector (210 nm).

Results are presented in FIG. 3. TMB3206 was able to convert furfuraland HMF in approximately 5 hours, whereas HMF was still present in themedium 20 hours after hydrolysate addition in the experiment with thecontrol strain TMB3280. TMB3206, which had 5 times higher HMF uptakerate, showed a more or less constant CER (FIG. 3B), while CER graduallydecreased for the control strain (FIG. 3A). The specific ethanolproductivity was also lower for the control strain compared with TMB3206overexpressing the mutated ADH1 gene.

Example 5 Changing Cofactor Balance and Product Formation in a StrainExpressing NADH-Dependent HMF Reductase and the Pichia stipitis XylosePathway

Overexpression in S. cerevisiae TMB3320

Plasmid YEplac-HXT-ADH1-S110P-Y295C encoding a mutated ADH1 gene (asdescribed in Table 4), as well as the corresponding empty plasmidYEplacHXT (Karhumaa et al., 2005, Investigation of limiting metabolicsteps in the utilization of xylose by recombinant Saccharomycescerevisiae using metabolic engineering. Yeast. 22(5):359-368) were usedto transform the xylose consuming strain S. cerevisiae TMB3320 resultingin strains TMB3291 and TMB3290, respectively. TMB3320 was obtained bytransformation of TMB3043 (leu2, ura3) with the plasmid YIplac128-XRXDH,which directs the expression of xylose reductase and xylitoldehydrogenase from Pichia stipitis. As expected, NADH-dependent HMFreductase activity was only detected in the strain carrying the mutatedADH1 gene (data not shown).

Changes in Cofactor Balance in Anaerobic Continuous Culture with HMF

Strains TMB3291 and TMB3290 were pre-grown overnight at 30° C. and 200rpm in 1 L—shake-flasks with 100 ml defined medium (Verduyn et al.,1992. Effect of benzoic acid on metabolic fluxes in yeasts: acontinuous-culture study on the regulation of respiration and alcoholicfermentation. Yeast, 8, 501-517) supplemented with 20 g/l glucose. Thesame defined medium supplemented with ergosterol (0.075 g/l) and Tween80 (0.84 g/l), with or without HMF (2 g/l), was used in subsequentcontinuous culture experiments. The starting OD620 in the fermentor was0.2. Fermentation was carried out in 400 mL media at 30° C. in BelachBiotech fermentors with dilution rates of 0.06 h⁻¹ and 0.12 h⁻¹.Anaerobic conditions were maintained by continuously sparging 0.2litre/minute nitrogen gas. The stirring rate was 600 rpm and pH 5.5 wasmaintained with 0.75M NaOH.

Cell concentration was determined from dry-weight measurements fromduplicate samples. For dry-weight samples 5 ml of the cell suspensionwere vacuum filtered through pre-weighed Gelman filters (ø47 mmSupor-450, 0.45 μm). Filters were washed with water and dried. Samplesfor analysis of metabolite concentrations were taken at culturesteady-state, i.e. carbon dioxide production was constant afterapproximately 5 culture volume changes. The samples were filteredthrough 0.2 μm filters. The concentrations of glucose, xylose, xylitol,glycerol, acetate, ethanol and HMF were separated on an Aminex HPX-87Hcolumn (Bio-Rad, Hercules, Calif.) at 65° C. The mobile phase was 5 mMH₂SO₄ with a flow rate of 0.6 ml/min. All compounds were detected with arefractive index detector, except for HMF which was detected with aUV-detector at 210 nm.

The glucose and xylose consumption and product formation were similarfor both strains when no HMF was added to the feed-medium. In contrast,when adding HMF to the feed-medium, considerably changes occurred. Thexylitol yield decreased for the strain TMB3291 (ADH1-S110P-Y295C) whileit increased for TMB3290 strain (control). The glycerol yields were 50%less for the ADH1-S110P-Y295C strain, while it was not affected in thecontrol strain. Furthermore, biomass yields of ADH1-S110P-Y295C strainincreased in presence of HMF. Acetate yields increased for both strains.With the highest dilution rate (0.12 h⁻¹) xylose consumption decreased50% for control strain but was not affect in ADH1-S110P-Y295C strain.Furthermore, half of the added HMF in the feed was still present in thefermentor with control strain, but it was completely converted in thefermentor with ADH1-S110P-Y295C strain. Ethanol yields for both strainswere not affected by the addition of HMF and were the same for bothstrains.

The experiment confirmed that increased in vivo NADH-dependent HMFreduction changes cofactor balance and consequently by-productdistribution in S. cerevisiae strains.

Example 6 Improved Ethanol Production in a Strain ExpressingNADH-Dependent HMF Reductase and the Pichia stipitis Xylose PathwayAnaerobic In Vivo HMF Reduction

Inoculum cultures of strains TMB3290 (control) and TMB3291(overexpressing the mutated ADM-S110P-Y295C gene) were grown overnightat 30° C. and 150 rpm in 1 L—shake-flasks with 100 ml of 2 timesconcentrated defined mineral medium (as described in Verduyn et al.,1992, Effect of benzoic acid on metabolic fluxes in yeasts: acontinuous-culture study on the regulation of respiration and alcoholicfermentation. Yeast 8, 501-517) supplemented with 40 g/l glucose and 200ml/l phtalate buffer (10.2 g/L KH phtalate, 2.2 g/l KOH). The samedefined medium with or without HMF (2 g/l), was used in subsequent batchexperiments. The medium was supplemented with 20 g/l glucose, 50 g/lxylose, ergosterol (0.075 g/l) and Tween 80 (0.84 g/l). The startingOD620 in batch fermentation was 0.5. Fermentation was carried out in 1 Lmedia at 30° C. in Braun Biotech fermentors. pH 5.5 was maintained with3M KOH. The stirring rate was 200 rpm.

A representative experimental result for the two strains in the presenceand absence of HMF is shown in FIG. 4.

The glucose and xylose consumption and product formation were similarfor both strains when no HMF was added to the medium (data not shown).In presence of HMF, the control strain (TMB3290) consumed glucose andxylose slower than the ADH1-S110P-Y295C strain (TMB3291)(FIG. 4).Furthermore, at the end of the fermentation the ADH1-S110P-Y295C strainhad consumed twice as much xylose as the control strain, which resultedin higher final ethanol concentration. The results support that the HMFconversion is faster in ADH1-S110P-Y295C strain (FIG. 4).

Example 7 Kinetic Characterization of ADH1 Mutants

Plasmids carrying individual or combined mutations within the ADH1 genegenerated by site directed mutagenesis in Example 3 were introduced instrain BY4741by transformation according to the lithium acetate method(Gietz et al., 1992, Improved method for high efficiency transformationof intact yeast cells. Nucleic Acids Research 20:1425). Selection wasperformed on SD plates without uracil. The resulting strains and thecorresponding mutations are summarized in Table 5.

Cells were grown aerobically at 30° C. in 25 mL SD medium without uracil(in 250 mL baffled flask). Cell extracts were prepared by harvesting thecells at OD620 7.5-8.5, re-suspending the cells in Y-PER solution(Pierce, Rockford, Ill., USA), 1 mL/0.6 gr cells, shaking at roomtemperature for 50 min. The supernatant was collected aftercentrifugation for 20 min at 15000 g.

In vitro NADH-dependent furan reductase activity was measured byfollowing the oxidation of NADH at 340 nm in a Hitachi U-2000spectrophotometer (Hitachi, Tokyo, Japan). NADH-dependent aldehydereduction was determined (Wahlbom and Hahn-Hägerdal, 2002) with 200 μMNADH. Reduction kinetics were determined for acetaldehyde (range500W-100 mM), furfural (100 μM-20 mM) and HMF (500 μM-20 mM). For nativeAdh1 furfural was used up to 40 mM.

Modeling was performed according to the Michaelis-Menten equation withthe addition of a substrate inhibition constant:V=(Vmax·[S])/(Km+[S]+[S]2/KI), V—velocity, Vmax—maximal velocity,Km—affinity constant, Ki—substrate inhibition constant, and[S]—substrate concentration. Parameter value estimation was according tothe least square method, using the solver function in Microsoft® Excel2002. HMF activity parameters of strain BY474-ADH1-S117L were estimatedwithout the substrate inhibition constant due to a clear discrepancy ofthe substrate inhibition model with actual data.

The results are summarized in table 6. The improved values for Km andVmax of the different mutants clearly demonstrated the importance ofmutation Y295C for HMF and furfural reduction. Comparison of kineticparameters of the strains BY474-ADH1-S117L andBY4741-ADH1-110P-L117S-295C showed that L in position 117 is beneficial,since it significantly increases activities towards both furaldehydes.

TABLE 5 Strains and amino-acid differences among different Adh1 variantsStrain Position Position 59 110 117 148 152 295 BY4741-control V S L Q IY BY4741-ADH1-S110P-Y295C V P L Q I C BY4741-ADH1-Y295C T S L E V CBY474-ADH1-S117L T P L E V C BY4741-ADH1-110P-L117S-295C T P S E V C

TABLE 6 Enzyme kinetics of five Adh1 variants against differentsubstrates. Kinetic parameters were established with cell extracts ofΔAdh1 strain (BY4741) with the respective ORF overexpressed. BY4741-BY4741- Adh1 BY4741- ADH1-S109P- ADH1- BY474-ADH1- BY4741-ADH1- variantcontrol Y294C Y294C S116L 109P-L116S-294C HMF Vmax n.d 11090 36406320**    3120 (mU)/mg Km (mM) 9.45 13.1 4.28 4.3 Ki (mM) 20.8 27.3 n.d9.82 [S]_(Vmax,) 10-20 (4560) 20 (1550) 10 (4900) 5-10 (1320)(obsV_(max))* RSQ 0.999 0.98  0.975 0.984 Furfural Vmax 6530 5657 779211700     1625 (mU)/mg Km (mM) 18.8 0.13 0.34 0.33 0.04 Ki (mM) 13631.32 6.66 4.4  1.07 [S]_(Vmax,) 30 (4345) 0.25-0.5 (3480)   1 (5320)  1(7600) 0.25 (1230) (obsV_(max))* RSQ 0.97 0.984 0.986  0.992 0.974Acetaldehyde Vmax 45070 16509 32455 23060     4736 (mU)/mg Km (mM)0.216899 0.83 3.03 1.74 1.69 Ki (mM) 12.5326 46 56 96    49 [S]_(Vmax,) 1 (35300)    5 (14300)  10 (22000) 10-25 (17600)     10 (3700)(obsV_(max))* RSQ 0.987 0.97 0.974  0.979 0.968 *[S]_(Vmax) - substrateconcentration (mM) at observed V_(max) (obsV_(max), mU/mg) **V_(max) andK_(m) values derived from a model without substrate inhibition factor,see text for details n.d. - not detected

1. An isolated polypeptide having NADH dependent HMF reductase activity,wherein said polypeptide shows 80% homology to the amino acid sequenceshown in SEQ ID NO:2 and which differs from SEQ ID NO:2 in that at leastS 117L and Y295C or Y295S or Y295T or S1 1OP are substituted.
 2. Theisolated polypeptide according to any of claim 1, wherein saidpolypeptide differs from SEQ ID NO:2 in that S 117L and Y295C and S1 1OPare substituted.
 3. The isolated polypeptide according to claim 1,wherein said polypeptide has at least 90% homology to SEQ ID NO:2. 4.The isolated polypeptide according to claim 3, wherein said polypeptidehas at least 95% homology to SEQ ID NO:2.
 5. The isolated polypeptideaccording to claim 4, wherein said polypeptide has at least 98% homologyto SEQ ID NO:2.
 6. The isolated polypeptide according to claim 5,wherein said polypeptide has at least 98.5% homology to SEQ ID NO:2. 7.The isolated polypeptide according to claim 6, wherein said polypeptidehas at least 98.7% homology to SEQ ID NO:2.
 8. The isolated polypeptideaccording to claim 7, wherein said polypeptide has at least 98.8%homology to SEQ ID NO:2.
 9. The isolated polypeptide according to claim8, wherein said polypeptide has at least 99% homology to SEQ ID NO:2.10. A nucleotide sequence encoding the polypeptide according to claim 1.11. A vector comprising a nucleotide sequence as defined in claim 10.12. A host cell comprising a nucleotide sequence as defined in claim 11or a vector as defined in claim
 11. 13. The host cell according to claim12, wherein said host cell is selected from the group consisting ofeukaryotic and prokaryotic cells.
 14. The host cell according to claim13, wherein said cells are selected from the group consisting ofbacteria, yeast and fungi.
 15. The host cell according to claim 14,wherein said bacteria is selected from the group consisting ofEscherichia coli, Klebsiella sp. and Zymomonas mobilis and said fungi isfilamentous fungi.
 16. The host cell according to claim 14, wherein saidyeast cell is selected from the group Saccharomyces sp.,Schizosaccharomyces sp., Kluyveromyces sp., Pichia sp., Hansenula sp.,Candida sp., and Yarrowia sp.
 17. The host cell according to claim 16wherein said host cell is selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces bay anus and Saccharomycescarlsbergensis.
 18. The host cell according to claim 17 wherein saidhost cell is Saccharomyces cerevisiae.
 19. Use of the polypeptide havingNADH dependent HMF reductase activity according to claim 1, in theproduction of bulk chemicals from lignocellulosic feedstock and furancontaining material.
 20. Use according to claim 19 wherein said bulkchemical is selected from the group consisting of ethanol, butanol,lactate, succinate, glycerol, niannitol, L-ascorbic acid, xylitol andhydrogen gas.